Base editing-mediated one-step inactivation of the Dnmt gene family reveals critical roles of DNA methylation during mouse gastrulation

During embryo development, DNA methylation is established by DNMT3A/3B and subsequently maintained by DNMT1. While much research has been done in this field, the functional significance of DNA methylation in embryogenesis remains unknown. Here, we establish a system of simultaneous inactivation of multiple endogenous genes in zygotes through screening for base editors that can efficiently introduce a stop codon. Embryos with mutations in Dnmts and/or Tets can be generated in one step with IMGZ. Dnmt-null embryos display gastrulation failure at E7.5. Interestingly, although DNA methylation is absent, gastrulation-related pathways are down-regulated in Dnmt-null embryos. Moreover, DNMT1, DNMT3A, and DNMT3B are critical for gastrulation, and their functions are independent of TET proteins. Hypermethylation can be sustained by either DNMT1 or DNMT3A/3B at some promoters, which are related to the suppression of miRNAs. The introduction of a single mutant allele of six miRNAs and paternal IG-DMR partially restores primitive streak elongation in Dnmt-null embryos. Thus, our results unveil an epigenetic correlation between promoter methylation and suppression of miRNA expression for gastrulation and demonstrate that IMGZ can accelerate deciphering the functions of multiple genes in vivo.

| The distribution of off-target mutations in the genome. a, The Manhattan plot shows the distribution of all off-target mutations of different groups (hA3A-eBE3-Y130F, Tyr, and Crygc) in the genome. The abscissa is the location of the mutations on the chromosome, and the ordinate is the frequency of each mutation. Each point presents a mutation (SNV or indel), and the different colors indicate different chromosomes. b, Representative Sanger sequence chromatograms show the verification results of non-synonymous off-target mutations in each group. Summary of the verification was shown in Supplementary Data 2. Black arrows indicate the mutant sites. All check primers are listed in Supplementary Data 9. All source data are provided as a Source Data file.

Supplementary Fig. 4 | One-step generation of Tet-TKO embryos by IMGZ.
a, Schematic of the procedure to design sgRNAs to target Tet2 using Base-Editor. Base-Editor website has been established to design sgRNAs of cytosine base editors to induce stop codons in a specific gene. Coded by C# ASP.NET. Gene name or Refseq ID is required for sgRNA designing. b, Sequence of sgRNAs designed by Base-Editor for targeting Tet1, Tet2, and Tet3, respectively. The PAM sequence and the intended mutant base are shown in red and green, respectively. c, d, e, Representative images of blastocysts obtained by injection of hA3A-eBE3-Y130F mRNA and different sgRNAs targeting Tet1 (c), Tet2 (d), and Tet3 (e), respectively, into zygotes carrying Oct4-EGFP transgene. Scale bar, 100 μm. Three independent experiments were analyzed for each group. f, Representative images of Tet1/2/3-TKO blastocysts. Green fluorescence indicates the expression of Oct4-EGFP. Scale bar, 50 μm. Three independent embryos were analyzed for each group. g, The editing efficiencies of mutant sites in three Tet1/2/3-TKO blastocysts detected by blunting cloning sequencing. All source data are provided as a Source Data file.

Supplementary Fig. 5 | Generation of Dnmt1-KO, Dnmt3a/3b-DKO, and Dnmt-
null embryos by the IMGZ system and mating. a, Sequence of sgRNAs designed by Base-Editor for targeting Dnmt1, Dnmt3a, and Dnmt3b, respectively. The PAM sequence and the intended mutant base are shown in red and green, respectively. b, Methylation analysis of H19-DMR, Snrpn-DMR, and Line1 promoter in Dnmt1-KO and Dnmt3a/3b-DKO embryos. DNA was extracted from E9.5 embryos. Open circles represent unmethylated CpG sites, whereas filled circles represent methylated CpG sites. c, Representative images of Dnmt1-KO and Dnmt3a/3b-DKO embryos at E9.5 and E10.5 generated by the IMGZ system. Three independent embryos were analyzed for each group. Scale bars, 500 μm in the up panel and 2 mm in the down panel. d, Representative images of Dnmt-null blastocysts and E6.5 embryos. Green fluorescence indicates the expression of Oct4-EGFP. Three independent embryos were analyzed for each group. Scale bar, 50 μm in the left panel and 100 μm in the right panel. e, Representative Sanger sequence chromatograms of one Dnmt-null blastocyst. 11 of 16 checked blastocysts show homozygous mutant chromatograms. f, The editing efficiencies of mutant sites in four Dnmt-null blastocysts detected by blunting cloning sequencing. g, Strategy of generating Dnmt-null embryos using germline-specific conditional knockout parents (Stra8-Cre and ZP3-Cre). h, Frequencies of 5mC modified nucleotides in the genomic DNA of wild-type (n = 3), Dnmt3a/3b-DKO (n = 7) and Dnmt-null (n = 6) embryos at E8.5 determined by quantitative mass spectrometry. Data are mean ± s.e.m for the indicated biological replicates. P-values were determined by Student's unpaired two-sided t-test. All source data are provided as a Source Data file.

Supplementary Fig. 6 | RNA-seq reveals impaired gastrulation-related pathways
in Dnmt-null embryos. a, Box plots show the expression of critical genes involved in some canonical signaling pathways related to gastrulation and primitive streak formation in mice, including BMP, Nodal, WNT, and FGF pathways, in Exe and Epi at E6.5 and E7.5. The central lines are the median of data. The lower and upper hinges correspond to the 25th and 75th percentiles. The end of the lower and upper whiskers are 1.5 * IQR (inter-quartile range). Data beyond the end of the whiskers are plotted as outliers. P-values were determined by unpaired two-sided t-test. b, Box plots show the expression of some lineage-specific markers, including primitive streak, ectoderm, mesoderm, and definitive endoderm, in Exe and Epi at E6.5 and E7.5. The central lines are the median of data. The lower and upper hinges correspond to the 25th and 75th percentiles. The end of the lower and upper whiskers are 1.5 * IQR (inter-quartile range). Data beyond the end of the whiskers are plotted as outliers. P-values were determined by unpaired two-sided t-test. All source data are provided as a Source Data file.
P-values were calculated by Deseq2. All source data are provided as a Source Data file.

Supplementary Fig. 8 | IMGZ-mediated generation of Dnmt/Tet mutant embryos.
a, Schematic diagram of sgRNAs used to construct Tet1/2/3;Dnmt1/3a/3b-6KO, Tet1/2/3;Dnmt1-4KO, and Tet1/2/3;Dnmt3a/3b-5KO embryos through the IMGZ system. b, Deep sequencing analysis of genome mutant sites of one control and three Tet/Dnmt-6KO embryos at E6.5, E7.5, and E8.5. The results indicated the conversion of CAG or CGG codon of Tet/Dnmt-6KO into TAG or TAA stop codon in resultant embryos. c, Representative images of control and Tet/Dnmt-6KO embryos at E4.0 and E6.5. Green fluorescence indicates the expression of Oct4-EGFP. Three independent embryos were analyzed for each group. Scale bars, 50 μm in the left panel and 200 μm in the middle panel. d, The ratio of epiblast area to the whole embryo in control (n = 6 for E6.5 and E7.5), Dnmt-null (n = 5 for E6.5 and n = 7 for E7.5), and Tet/Dnmt-6KO (n = 4 for E6.5 and E7.5) embryos at E6.5 and E7.5. Epiblast was indicated by the signal of Oct4-EGFP. Data are mean ± s.e.m of indicated biological replicates P-values were determined by Student's unpaired two-sided t-test. e, Representative images of RNA in situ hybridization of T probe in the control, Dnmt-null, and Tet/Dnmt-6KO embryos at E7.5, showing primitive streak elongation failure in these embryos. More than three independent embryos were analyzed for each group. Scale bar, 100 μm. f, The ratio of primitive streak area to the whole embryo of control (n = 7), Dnmt-null (n = 7), and Tet/Dnmt-6KO (n = 8) embryos at E7.5. The primitive streak was indicated by the signal of T. Data are mean ± s.e.m of indicated biological replicates. P-values were determined by Student's unpaired two-sided t-test. g, Representative images of control, Tet/Dnmt1-4KO, and Tet/Dnmt3a/3b-5KO embryos at E9.5 generated by the IMGZ system. Three independent embryos were analyzed for each group. Scale bar, 500 μm. All source data are provided as a Source Data file.   Exe and Epi at E6.5 and E7.5 related to Fig. 6b. E3.5 TE and E4.0 ICM data obtained from a previous study 2 show that DNA methylation has been deposited on these loci at the blastocyst stage. n, the number of overlapped elements. P-values were determined by Student's unpaired two-sided t-test. The central lines are the median of data. The lower and upper hinges correspond to the 25th and 75th percentiles. The end of the lower and upper whiskers are 1.5 * IQR (interquartile range). Data beyond the end of the whiskers are plotted as outliers. All source data are provided as a Source Data file. Supplementary Fig. 11 | Upregulation of retrotransposons and retrotransposonrelated genes in Dnmt mutant embryos. a, Box plots show CG methylation levels of retrotransposons in Exe and Epi with different mutations at E6.5 and E7.5 measured by WGBS, including LTR retrotransposons (long terminal repeats) and non-LTR retrotransposons. The central lines are the median of data. The lower and upper hinges correspond to the 25th and 75th percentiles. The end of the lower and upper whiskers are 1.5 * IQR (inter-quartile range). Data beyond the end of the whiskers are plotted as outliers. P-values were determined by unpaired two-sided t-test. ERVs, endogenous retroviruses; LINEs, long interspersed elements; SINEs, short interspersed elements; the Alu sequences are named based on sharing a common cleavage site for the AluI restriction enzyme. b, Expression of retrotransposon families, including ERV1, ERVK, ERVL, LINE1, LINE2, SINE, and Alu, in Exe and Epi at E6.5 and E7.5 measured by RNA-seq, indicating that ERVK was significantly up-regulated in Dnmt mutant embryos (fold change > 2). c, Expression of all genes, genes located close to all LTRs (± 20kb), and genes located close to up-regulated LTRs in Dnmt-null (compared to both Dnmt1-KO and Dnmt3a/3b-DKO). The central lines are the median of data. The lower and upper hinges correspond to the 25th and 75th percentiles. The end of the lower and upper whiskers are 1.5 * IQR (inter-quartile range). Data beyond the end of the whiskers are plotted as outliers. P-values were calculated between the up-regulated LTR groups and All-LTR group by the two-sided Wilcoxon test. The exact P-values were listed in Source Data file. All source data are provided as a Source Data file.